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研究生:邱蕙瑄
研究生(外文):Huei-Hsuan Chiu
論文名稱:由造成肝膿瘍之克雷伯氏肺炎桿菌分離一高抗原性分泌蛋白質
論文名稱(外文):Isolation of an Immunodominant Protein Secreted by Klebsiella pneumoniae Strains Causing Liver Abscess
指導教授:王錦堂王錦堂引用關係
指導教授(外文):Jin-Town Wang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:83
中文關鍵詞:肝膿瘍高抗原性克雷伯氏肺炎桿菌分泌蛋白質
外文關鍵詞:Klebsiella pneumoniaeliver abscessimmunodominantsecreted protein
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克雷伯氏肺炎桿菌是院內感染常見的致病菌。近二十年來,臺灣出現一種新型的侵襲性克雷伯氏肺炎桿菌原發性肝膿瘍感染,並有逐年增加的趨勢。我們以感染克雷伯氏肺炎桿菌而造成肝膿瘍的恢復期病人血清,偵測由細菌培養液收取而來的分泌蛋白質,發現一個具高抗原性的標的蛋白質。隨後以蛋白質體學的方法,先用二維電泳將此蛋白質分離,再用N端蛋白質定序、基質輔助雷射脫附游離/ 飛行時間質譜分析和液相層析質譜質譜分析等方法鑑定。最後,在液相層析質譜質譜分析的結果,得到標的蛋白質的部分胜肽序列,在和2003年年底剛完成的克雷伯氏肺炎桿菌臺大菌株NTUH-K2044完整基因體序列作比對之後,終於鑑定出此標的蛋白質的基因位於克雷伯氏肺炎桿菌大質體上第307個開放讀架(即KPP307)。KPP307的基因大小為921個鹼基對,轉譯成306個胺基酸、分子量大小為32 kDa的未知蛋白質。生物資訊學的分析結果顯示KPP307在N端有一段含25個胺基酸的訊息胜肽。我們經由蛋白質表現和西方墨點法重新確認標的蛋白質。另一方面,我們發現相較於健康人,在目前所有感染克雷伯氏肺炎桿菌而造成肝膿瘍的恢復期病人血清樣本中,利用西方墨點法皆可檢測到1:1000以上高效價的抗KPP307抗體,顯示此蛋白質在診斷侵襲性克雷伯氏菌感染上的潛在價值。然而,KPP307在侵襲性克雷伯氏肺炎桿菌致病機轉上所扮演的角色為何則有待進一步研究。
Klebsiella pneumoniae is a common pathogen, causing nosocomial infections. Over the past two decades, a new type of invasive K. pneumoniae disease has emerged in Taiwan that typically presents a community-acquired primary liver abscess. In this study we attempted to analyze immunodominant proteins secreted by K. pneumoniae strains causing liver abscess. We found a target protein from culture supernatants by immunoblot analyses with convalescent-phase patient sera. Subsequently we identified the protein by proteomic approach including 2DE, N-terminal protein sequencing, MALDI-TOF MS and LC-MS/ MS. LC-MS/ MS identified the target protein by comparing the partial peptide sequence with the full genome sequence of clinical strain NTUH-K2044. A novel 921-bp locus, KPP307, which encoded a 32-kDa unknown protein, was the 307th open reading frame located on the large plasmid of K. pneumoniae. Bioinformatic analyses revealed that KPP307 started with a signal peptide composed of 25 amino acids. All convalescent-phase patient serum samples collected so far contained Western-blot antibodies at a titer of 1:1000 or greater, indicating potential applications for this protein in diagnosis of invasive K. pneumoniae infections. However, the role of KPP307 in virulence mechanism awaits further studies.
摘要………………………………………………1
Abstract…………………………………………2
序言………………………………………………3
材料……………………………………….……..11
一、克雷伯氏肺炎桿菌菌株………………….…………………..........…………..11
二、大腸桿菌菌株………………………………….…………………..........……..11
三、血清……………………………….…………………………………...............12
四、抗體…………………………….……………………………………...............12
五、培養基………………………….………………………………………............13
六、抗生素………………………………….………………………………............13
七、質體……………………………….…………………………………...............13
八、引子…………………………………………………..………........………......14
九、限制酵素……………………………………........…………………………….14
十、試劑…………………………………………….……........……………….......15
十一、套組………………………………….........….…………………………......18
十二、設備與器材……………………………….........……………………………19
方法………………………………………………21
一、克雷伯氏肺炎桿菌之分泌蛋白質………………………........……………….21
1.1 克雷伯氏肺炎桿菌之培養…………………………………….........……….. 21
1.2 克雷伯氏肺炎桿菌分泌蛋白質之收取……………….……….........………..21
1.3 以Bradford method測定蛋白質濃度………………….…………..........…...22
二、克雷伯氏肺炎桿菌高抗原性分泌蛋白質之分離與篩選…….…………........23
2.1 SDS-聚丙烯醯胺膠體電泳……………………………………..........……......23
2.2 西方墨點法……………………………………………………………............24
2.3 二維膠體電泳…………………………………………………..........………..24
三、克雷伯氏肺炎桿菌高抗原性分泌蛋白質之鑑定…………………………….25
3.1 N端蛋白質定序前處理………………………………………...……….……...25
3.2 膠內酵解……………………………………………………..………….……..26
四、蛋白質表現與再確認………………………………………….........…..……..27
4.1 克雷伯氏肺炎桿菌基因體去氧核糖核酸的抽取……………….….........…..27
4.2 聚合酶連鎖反應…………………………………………….........…….……..28
4.3 洋菜膠電泳………………………………………………………..........……..29
4.4 去氧核糖核酸之分離與回收………………………………….........….……..29
4.5 勝任細胞的製備………………………………………………….….........…..30
4.6 TA選殖………………...…………………………………………..……..........30
4.7 以聚合酶連鎖反應挑選正確選殖株…………………………….…..….........31
4.8 質體的抽取……………………………………………………….……...........32
4.9 限制酵素酵解作用……………………………………………………............32
4.10 接合反應………………………………………………………………..........33
4.11 去氧核糖核酸定序…………………………………………………..............33
4.12 重組蛋白質的表現………………………………………….…….........……34
4.13 重組蛋白質純化…………………………………………….………….........34
4.14 標的蛋白質再確認-競爭阻礙作用…………………………………….........36
五、高抗原性蛋白質KPP307的生物資訊學分析……………….………….......36
六、病人血清中抗KPP307抗體的偵測………………………….………….......37
結果………………………………………………38
一、克雷伯氏肺炎桿菌之分泌蛋白質………………………………….........……38
二、克雷伯氏肺炎桿菌高抗原性分泌蛋白質之分離與篩選………….……........39
三、克雷伯氏肺炎桿菌高抗原性分泌蛋白質之鑑定………………….……........40
四、蛋白質表現與再確認…………………………….………………………........43
五、高抗原性蛋白質KPP307的生物資訊學分析…………………….…….......44
六、病人血清中抗KPP307抗體的偵測…………………….......………….……45
討論………………………………………………47
圖表………………………………………………55
圖一、克雷伯氏肺炎桿菌分泌蛋白質以SDS-聚丙烯醯胺膠體電泳分析,經CBR染色後的型態。…………………………………….........................………55
圖二、克雷伯氏肺炎桿菌分泌蛋白質經一維SDS-聚丙烯醯胺膠體電泳分析後,以西方墨點法偵測具高抗原性之蛋白質。…….……....................………56
圖三、克雷伯氏肺炎桿菌分泌蛋白質經二維膠體電泳分析後,以西方墨點法偵測具高抗原性之蛋白質。……………………....................………….………57
圖四、克雷伯氏肺炎桿菌分泌蛋白質以二維膠體電泳分析,經CBR染色後的型態。………………………....................……………………….……………58
圖五、KPP307之MALDI-TOF MS質量圖譜。……............……..……………59
圖六、KPP307的DNA序列。…………………………….........…………..……60
圖七、建構重組KPP307表現載體。………………..………….........…….……61
圖八、重組KPP307的蛋白質表現,經SDS-聚丙烯醯胺膠體電泳分析及CBR染色後的結果。…………………………………..................….……..……62
圖九、純化之重組KPP307在一維SDS-聚丙烯醯胺膠體電泳分析後,以病人血清及利用競爭阻礙作用進行西方墨點法的結果,再次證明KPP307為標的蛋白質。………………………….……………..............................……63
圖十、克雷伯氏肺炎桿菌分泌蛋白質KPP307在一維SDS-聚丙烯醯胺膠體電泳分析後,以競爭阻礙作用進行西方墨點法的結果,再次證明KPP307為標的蛋白質。………………………….………...........................…………64
圖十一、重組KPP307在一維SDS-聚丙烯醯胺膠體電泳分析後,以西方墨點法偵測在病人血清中的抗KPP307抗體。…………....................…………65
圖十二、重組KPP307蛋白質表現後,以thrombin protease切下GST,經SDS-聚丙烯醯胺膠體電泳分析及CBR染色的結果。…......................………66
表一、二維膠體電泳資料。…………………………………..............……………67
表二、KPP307以trypsin酵解後胜肽質量之MALDI-TOF MS實測值與利用ExPASy網站計算之預測值比較。………………….……............………68
參考文獻…………………………………………70
附錄………………………………………………76
一、利用ExPASy網站預測KPP307之胜肽質量………….…….......…………76
二、利用SMART網站分析KPP307之結構…………………..……….......……79
三、利用NCBI網站之BLAST程式比對的結果……………………………........81
3.1 和KPP307有相似性的基因…………………………………………….........81
3.2 和KPP307有相似性的蛋白質………………………………………….........83
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